Methods for Assessing Risk for Cardiac Dysrythmia in a Human Subject

ABSTRACT

The present invention relates to methods for assessing the risk of a patient for developing a potentially fatal cardiac dysrhythmia and for diagnosing Andersen&#39;s Syndrome. A tissue sample from a patient is obtained and the DNA or proteins of the sample isolated. From the DNA and protein isolates the sequence of the KCNJ2 gene or the Kir2.1 polypeptide can be obtained. The KCNJ2 gene or the Kir2.1 can be screened for alteration as compared to the wile-type sequence. An alteration in a copy of the KCNJ2 gene or a Kir2.1 polypeptide indicates that the patient has a high risk for developing a cardiac dysrhythmia and can be diagnosed with Andersen&#39;s Syndrome. The invention also related to isolated nucleic acid molecules with one or more alterations as compared to the wild-type sequence.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of prior filed U.S.application Ser. No. 10/475,452 filed Oct. 21, 2003, which claimspriority to U.S. Provisional Application Ser. No. 60/286,146 filed Apr.24, 2001. This application hereby incorporates by reference both suchapplications.

BACKGROUND OF THE INVENTION

1. The Field of the Invention

The present invention relates to methods of detecting risk for cardiacdisease. More specifically, the present invention relates to geneticbased methods for detecting a risk for a cardiac dysrhythmia in apatient and for diagnosing Andersen's Syndrome.

2. The Relevant Technology

Andersen's Syndrome (AS) is a rare disorder characterized by periodicparalysis, cardiac arrhythmias, and dysmorphic features. Canun, S., etal. (1999) Am J Med Genet. 85: 147-56; Sansone, V. et al. (1997) AnnNeurol 42: 305-12; Tawil, R. et al. (1994) Ann Neurol 35: 326-30. Thedysmorphology includes short stature, scoliosis, clinodactyl), wide-seteyes, small or prominent ears that are low set or slanted, a small chin,cleft pallet, and broad forehead. AS occurs either sporadically or as anautosomal dominant trait. In AS families, expression of thecharacteristic traits is highly variable. Thus, it is likely that the ASprotein plays a complex role in development and cell excitability withsome redundancy with other proteins.

The periodic paralyses and nondystrophic myotonias are a group of muscledisorders manifest by abnormal muscle relaxation (myotonia). Thismyotonia results from muscle hyperexcitability that sometimestransitions to inexcitability resulting in episodic weakness. Ventaculartachydysrhythmias are analogous to myotonia of skeletal muscle in thathyperexcitability leads to an abnormal, albeit highly organized, seriesof heart contractions that can also transition to inexcitability thusleading to death from cardiac dysrhythmias. The electrophysiologicalfeatures of such diseases suggest an underlying defect in membraneexcitability. Approximately 300,000 Americans die of cardiacdysrhythmias each year. Kannel, W. B. et al. (1987) Am Heart J 113,799-804; Willich, S, N. et al. (1987) Am J Cardiol 60: 801-6.

Many of the persons who die of cardiac dysrhythmias do not exhibit heartproblems prior to death. The fatal cardiac dysrhythmia may be triggeredby aerobic exercise such as running Thus, a patient may be at risk forcardiac dysrhythmias without exhibiting risk factors and without knowingto avoid certain types of activities or exercise. Moreover, certainmedications are known to induce cardiac dysrhythmias in patients withheart conditions. However, when a patient does not exhibit any of thefactors which would indicate a risk for cardiac dysrhythmias prior to adeadly episode, medical professionals cannot know what drugs to avoidprescribing to a patient.

Sudden Infant Death Syndrome (SIDS) is the leading cause of death ininfants between 1 month and 1 year of age. Most SIDS deaths occur when ababy is between 1 and 4 months of age. SIDS is the medical term used todescribe the sudden death of an infant under one year of age thatremains unexplained after a complete investigation, which includes anautopsy, examination of the death scene, and review of the symptoms orillnesses the infant had prior to dying and any other pertinent medicalhistory. A precise cause of SIDS is not known. However, sleep-inducedarrhythmias may be a factor in SIDS. It has been hypothesized thatchanges in the activity of the autonomic nervous system during sleepcould precipitate an arrhythmia resulting in sudden death.

The electrical properties of excitable tissues such as skeletal muscle,heart and neurons are determined, in part, by a number of ion channelsthat work in concert to provide properties appropriate for the functionof each tissue. The first ion channel mutations which were shown tocontribute to an episodic disorder were characterized about decade agowhen mutations in SCN4A, which encodes a voltage-gated sodium channel,were shown to cause hyperkalemic periodic paralysis. Ptacel, L. J. etal. (1991) Cell 67: 1021-7; Rojas, C. V. et al. (1991) Nature 354:387-9. This rare muscle disease formed the basis of the growing groupnow known as the channelopathies and led to predictions that cardiacdysrhythmias and epilepsies would be caused by mutations in homologousgenes. Ptacek, L. J. et al. (1991) Cell 67: 1021-7. Similarities betweenthese different episodic disorders suggested similar molecular bases ofthese disorders. The occurrence of both periodic paralysis and long QT(LQT) in Andersen's Syndrome strongly supports this hypothesis. Tawil,R. et al. (1994) Ann Neurol 35: 326-30. Since this initial discovery,periodic paralysis has been associated with mutations in voltage-gatedK.sup.+, Na.sup.+, Ca.sup.2+, and Cl.sup.− channels, while LQT has beenassociated with mutations in voltage-gated K.sup.+ and Na.sup.+channels. Jen, J. & Ptacek, L. J., METABOLIC AND MOLECULAR BASES OFINHERITED DISEASE, pp. 5223-5238(C. R. Scriver et al., McGraw-Hill,2001); Sanguinetti, M. (2001) Cell 104:569-580. To date, no humandisorders involving cardiac and skeletal muscle have been attributed tomutations in inward rectifying K.sup.+ channels.

Inward rectifier K.sup.+ channels (Kir) play a role in controlling cellexcitability and resting membrane potential in many different tissuesincluding heart, brain, and skeletal muscle. Doupnik, C. A. et al.(1995) Curr Opin Neurobiol 5: 268-77; Jan, L. Y., & Jan, Y. N. (1997) JPhysiol 505:267-82; Nichols, C. G., & Lopatin, A. N. (1997) Annu RevPhysiol 59, 171-91. Generally, Kir channels contribute to the finalrepolarization phase of cardiac action potentials by passing smallamounts of K.sup.+ out of the cell and bringing the membrane potentialback to resting membrane potential (E.sub.m). Structurally, Kir channelsresemble voltage-gated K.sup.+ channels; however, they are missing thefour N-terminal transmembrane domains including the S4 voltage sensor.Kir channels consist of an intracellular N-terminal domain, twotransmembrane segments M1 and M2) flanking a pore region, and anintracellular C-terminal segment; M1 and M2 correspond to S5 and S6 ofvoltage-gated channels. Kir subunits are believed to form either homo-or heterotetramers. Yang, J. et al. (1995) Neuron 15: 1441-7.

Kir2.1 (IRK1), encoded by the gene KCNJ2, is a member of the Kir2.xfamily of inward rectifying K.sup.+ channels expressed predominantly inheart, brain, and skeletal muscle. Kubo, Y. et al (1993) Nature 362,127-33 Raab-Graham, K. F. et al. (1994) Neuroreport 5, 2501-5. Thefunction of Kir2.1 has been studied primarily in the heart. It isclassified as a strong inward rectifier, that is, almost no currentpasses through these channels at potentials positive to −40 mV. Thus,strong inward rectification prevents excess loss of K.sup.+ during theplateau phase of the cardiac action potential, but allows outwardK.sup.+ flux during terminal repolarization and diastolic phases of theaction potential. Sanguinetti, M. C., & Tristani-Firouzi, M., CARDIACELECTROPHYSIOLOGY: FROM CELL TO BEDSIDE, pp. 79-86., (D. P. Zipes, & J.Jalife, eds., W. B. Saunders, 2000). Much less is known about the roleof Kir2.1 in other tissues such as the brain and skeletal muscle. It islikely that the role of Kir2.1 in skeletal muscle and neurons is similarto its role in the heart by controlling the resting membrane potentialand the terminal repolarization phase of the action potential.Interestingly, there is some evidence suggesting that Kir2.1 has somefunctional significance outside of modulating the action potential ofneurons and myocytes. Kir2.1 knockout mice have a complete cleft of thesecondary palate and a slight narrowing of the maxilla. Zaritsky, J. J.et al. (2000) Circ Res 87: 160-6. In rat, Kir2.1 mRNA is present byembryonic day 12 in bone associated structure of the head, limb, andbody. Karschin, C., & Karschin, A. (1997) Mol Cell Neurosci 10, 131-48.These findings provide some evidence for an underlying developmentalfunction of Kir2.1.

Currently the genetic cause of Andersen's Syndrome is not known.Moreover, persons with Andersen's Syndrome have a high risk for cardiacdysrhythmias. Accordingly it would be an advancement in the art toprovide a gene responsible for the Andersen's Syndrome phenotype. Itwould be a further advancement to provide a diagnostic test forAndersen's Syndrome. It would be a further advancement to provide amethod for detecting a risk for cardiac dysrhythmias in a patient.

SUMMARY OF THE INVENTION

The method of the present invention has been developed in response tothe present state of the art, and in particular, in response to theproblems and needs in the art that have not yet been fully solved by thecurrent state of medicine and genomics.

The present invention relates to a method of determining whether apatient has a heightened risk for a cardiac dysrhythmia. The methodemploys a genetic screen to determine if the patient has an alterationin single copy of the KCNJ2 gene. If the patient has an alteration in aKCNJ2 gene, the patient may have a heightened risk for a cardiacdysrhythmia as compared to a person without an alteration in his or hercopies of the KCNJ2 gene. An alteration in a copy of the KCNJ2 gene maybe a missense mutation, a deletion, an in-frame deletion, an insertion,or other mutation.

The screening of the patient's genome for alterations in the KCNJ2 genemay be accomplished in a variety of ways. For example, a tissue samplemay be obtained from the patient. One or both copies of the patient'sKCNJ2 gene may be isolated from the tissue sample, and one or bothcopies of the patient's KCNJ2 gene may be sequenced. The sequence of thepatient's KCNJ2 gene may then be compared to the sequence of a wild-typeKCNJ2 gene. A difference in the patient's KCNJ2 gene sequence comparedto the wild-type sequence indicates an alteration in the patient's KCNJ2gene, and it may be determined that the patient has a heightened riskfor a cardiac dysrhythmia.

Another way of detecting an alteration in the patient's KCNJ2 gene mayinclude comparing the patient's KCNJ2 sequence with the sequences ofknown mutations. For example, a copy of one or both of the patient'sKCNJ2 genes may be obtained and sequenced. The sequence of the patient'sKCNJ2 gene may then be compared to a library of sequences of KCNJ2 geneswith known mutations. If the sequence of the patient's KCNJ2 isidentical to one of the sequences with a known mutation, then it may bedetermined that the patient has an alteration in a copy of his or herKCNJ2 genes. Currently, fourteen mutations in KCNJ2 genes have beenisolated and sequenced from individuals and kindreds diagnosed withAndersen's Syndrome.

A probe may also be used to determine whether a patient has analteration in a copy of his or her KCNJ2 gene. Such probes may contain aDNA sequence complementary to a portion of a KCNJ2 gene with a knownmutation. The ability of the probe to bind to a copy of the patient'sKCNJ2 gene may be tested under high stringency conditions. If the probecan bind to the patient's KCNJ2 gene, then it may be determined that thepatient has a significant risk for a cardiac dysrhythmia. Such probesmay be constructed using the portion of a KCNJ2 gene containing analteration.

KCNJ2 encodes the polypeptides of the inward rectifying ion channelKir2.1. The phenotypic characteristic of a person with Andersen'sSyndrome can be attributed to alterations in Kir2.1. Thus, an alterationin Kir2.1 may indicate an alteration in KCNJ2. Thus, the sequence of apatient's Kir2. 1 polypeptide may be determined and compared to the wildtype sequence. Any alterations in the Kir2.1 polypeptide indicate analteration in a KCNJ2 gene.

The invention also relates to methods of genetically diagnosingAndersen's Syndrome in a patient. The method for diagnosing Andersen'sSyndrome may use the same steps as determining whether a patient has ahigh risk for a cardiac dysrhythmia. An alteration in the KCNJ2 gene mayprovide for the positive diagnosis of Andersen's Syndrome in a patient.An alteration in a copy of the KCNJ2 gene may be a missense mutation, adeletion, an in-frame deletion, or an insertion.

As with the method of determining a patient's risk for cardiacdysrhythmias in the method of diagnosing Andersen's Syndrome, thescreening of the patient's genome for alterations in the KCNJ2 gene maybe accomplished in a variety of ways. For example, a tissue sample maybe obtained from the patient and the KCNJ2 gene isolated. The sequenceof the KCNJ2 genes may be compared to wild-type or mutant sequences todetermine whether the patient's sequence contains an alteration. Alsoprobes may be used to detect an alteration. Other detection methods mayinvolve the analysis of the Kir2.1 peptides encoded by KCNJ2.

In another aspect of the invention, a method of assessing a risk forhuman subject for Sudden Infant Death Syndrome (SIDS) is presented. Themethod may be used to screen an infant for the presence of an alterationin a copy of the KCNJ2 gene. Alternatively, a parent, or sibling of theinfant may be screened for an alteration in a copy of their KCNJ2 genes.If the infant, parent, or sibling is found to have an alteration theKCNJ2 gene, then the infant may be at higher risk for SIDS. Such alteredKCNJ2 genes which may place an infant at risk for SIDS may have thesequence of SEQ ID NO 1 as altered by on or more mutations selected fromthe group consisting of A440T, G658A, A874C, C880T, G1127T, G1132A,C635T, G881A, G1135A, C785T, C452G, G439A, a 6 nucleotide deletionbeginning with nucleotide 1167, and a 12 nucleotide deletion beginningwith nucleotide 512.

The screening for an alteration in a copy of the KCNJ2 gene may beaccomplished in any of a number of ways. Generally a tissue sample suchas blood, hair, oral swab, and the like will be taken for the screen.The KCNJ2 gene may be sequence and the sequence compared to a wild-typesequence or a sequence with a known alteration. Alternatively, theability of a DNA binding probe containing a known alteration to bind tothe KCNJ2 gene from the subject may be used. Also the Kir2.1 polypeptidemay be used to screen for an alteration in the KCNJ2 gene.

The present invention also relates to isolated and purified nucleicacids which code for Kir2.1 polypeptides with an alteration in thepolypeptide sequence. Such nucleic acids may be KCNJ2 genes with analteration such as a insertion, a deletion, a missense mutation. SomeKir2.1 polypeptides have been isolated, purified and sequenced. Forexample an altered Kir2.1 polypeptide may have the sequence of SEQ IDNo. 2 as altered by one or more alterations selected from the groupconsisting of D71V, G144S, N216H, R218W, G300V, V302M, .DELTA.314-315,95-98, S136F, R218Q, E303K, P186L, T75R, and D71N. Nucleic acidmolecules that encode for the altered Kir2.1 polypepetide may be alteredKCNJ2 genes having a sequence of SEQ ID NO 1 as altered by one or moremutations selected from the group consisting of A440T, G658A, A874C,C880T, G1127T, G1132A, C635T, G881A, G1135A, C785T, C452G, G439A, a6nucleotide deletion beginning with nucleotide 1167, and a 12 bydeletion beginning with nucleotide 512. Such altered KCNJ2 genes may beinserted into a vector or a cell for use in studies of the function ofthe Kir2.1 ion channel and the like.

SEQUENCE LISTING

The sequence listing attached, in disc format is hereby incorporated byreference in this application (See attached disc labeled “SequenceListing.txt” created Oct. 12, 2007 that is 16 KB). There is no newmatter on the disc.

BRIEF DESCRIPTION OF THE DRAWINGS

A more particular description of the invention briefly described abovewill be rendered by reference to specific embodiments thereof which maybe understood by reference to the appended figures. The figures relateto only typical embodiments of the invention and are not therefore to beconsidered to be limiting of its scope. The invention will be describedand explained with additional specificity and detail through the use ofthe accompanying drawings in which:

FIG. 1A is a graphic representation of the pedigree of kindred 4415exhibiting variable expressivity and associated KCNJ2 mutation. Femalesare denoted with circles and males with squares. An “*” denotes anindividual that was not included in the genome-wide linkage screen.

FIG. 1B is a graph illustrating a sequence chromatograph of an affectedindividual with an A to T transversion corresponding to the D71Vmutation.

FIG. 1C depicts the amino acid alignment of one subunit from each of theseven members of the inward rectifying K.sup.+ channels. The mutation isdenoted above the alignment. Lowercase letters denote conservative aminoacid changes, whereas a “.” denotes anon-conservative amino acid change.

FIGS. 2A and 2B depict the structure of Kir2.1 in relationship tovoltage-gated K.sup.+ channels. In FIG. 2A the structure ofvoltage-gated K.sup.+ channels is shown. The S4 voltage-sensing segmentis denoted with “+'s”. In FIG. 2B the structure of inward rectifyingK.sup.+ channels is shown. The locations of identified mutations arerepresented on the structure.

FIG. 3 depicts a map of the Andersen's Syndrome locus. Markers D175787and D175784 are the proximal and distal recombinant boundariesrespectively. Distance is marked between each recombinational boundariesand D175949. The location of three candidates is shown to the right.

FIG. 4 shows the pedigree of additional AS kindreds with identifiedmutations in Kir2.1. Females are denoted with circles and males withsquares. The kindred number and mutation are denoted above eachpedigree. Families in which the first generation is marked as“unaffected”0 represent de novo mutations. “Uncertain” individuals arethose for whom there was no clinical data.

FIG. 5 is a series of graphs illustrating sequence chromatography ofaffected AS individuals. Mutations of Kir2.1 in Andersen's patientsoccur in highly conserved residues. Chromatographs of the nucleotidesequence corresponding to each mutation are shown. “*” indicates thatthe nucleotide sequence shown is the reverse complement of codingsequence. Below each chromatograph is an alignment of the first memberof all human Kir families. Mutant residues are denoted above eachalignment. “.DELTA.” marks a deletion of the residue below it. Lowercaseletters denote conservative amino acid changes, whereas a “.” denotes anon-conservative amino acid change.

FIGS. 6A through 6C depict the functional effects of D71V Kir2.1mutation. FIG. 6A depicts WT Kir2.1 current elicited by 200 ms stepdepolarizations from −150 to −10 mV, from a holding potential of −70 mV.FIG. 6B depicts currents induced by injection of oocytes with H.sub.20alone, D71V Kir2.1 and co-injection of D71V and WT Kir2.1. Smallendogenous currents were recorded following injection of H.sub.20 orD71V. Co-injection of D71V and WT Kir2.1 resulted in inwardly rectifyingK.sup.+ currents. Note the smaller scale axis, compared to A. FIG. 6Cshows instantaneous current-voltage relationships for oocytes injectedwith WT (filled squares), D71V (open triangles) and co-injected WT andD71V (filled circles) Kir2.1. Data represent mean.+−.SEM, n=8-10 oocyteseach group.

FIGS. 7A through 7C depicts the functional effects of R218W Kir2.1mutation. FIG. 7A shows WT Kir2.1 current elicited by the voltageprotocol as shown. FIG. 7B depicts currents induced by injection ofoocytes with H.sub.20 alone, R218W Kir2.1 and co-injection of R218W andWT Kir2.1. Small endogenous currents were recorded following injectionof H.sub.20 or R218W. Co-injection of R218W and WT Kir2.1 resulted ininwardly rectifying K.sup.+ currents whose amplitude was larger thanthat induced by co-injected D71V and WT (see FIG. 6). FIG. 7Cillustrates instantaneous current-voltage relationships for oocytesinjected with WT (filled squares), 1/2 WT (down triangles), R218W (uptriangles) and co-injected WT and R218W (filled circles) Kir2.1. Oocyteswere injected with 23 ng total cRNA, with the exception of 1/2 WT whichwas injected with 11.5 ng WT cRNA. Currents induced by injection of 11.5ng WT were approximately one-half that induced by 23 ng WT Kir2.1. Datarepresent mean.+−.SEM, n=8-10 oocytes each group.

DETAILED DESCRIPTION OF THE INVENTION

As discussed previously, Andersen's Syndrome is a rare disordercharacterized by periodic paralysis, cardiac arrhythmias, and dysmorphicfeatures. In the past Andersen's Syndrome has been diagnosed based onthe phenotypic expression of the dysmorphic features, paralysis, andcardiac arrhythmias. AS occurs either sporadically or as an autosomaldominant trait. In AS families, expression of the characteristic traitsis highly variable. Thus, AS may seemingly skip a generation because ofthe low level of phenotypic expression or non-penetrance in a personwithin the AS kindred. It is likely that the AS protein plays a complexrole in development and cell excitability with some redundancy withother proteins.

The invention is based on the discovery that mutations in the Kir2.1gene, KCNJ2, cause the triad of phenotypes in Andersen's Syndrome,including periodic paralysis, cardiac arrhythmias, and dysmorphicfeatures. Andersen's Syndrome mutations involve residues in importantfunctional domains. Several lines of evidence suggest that all of theidentified mutations may result in functional consequences for Kir2.1.First, whence-expressed with wild-type subunits in Xenopus oocytes, D71Vand R218W has dominant negative effects on Kir2.1 current. Second,fourteen mutations in KCNJ2 have been identified, and all of theidentified mutations involve very highly conserved residues across allfamilies of the Kir subunits. Third, two mutations were identifiedwithin the pore helix, which is a region conserved across voltage- andnon-voltage-gated K.sup.+ channels. Specifically, mutations in the GYGsignature sequence of Kir channels lead to 1) a dominant negative effectand 2) a gain of function effect (Na.sup.+ influx) in the weaver mouse.Tinker, A. et al. (1996) Cell 87: 857-68; Slesinger, P. A. et al. (1996)Neuron 16: 321-31. The weaver phenotype of ataxia, tremor, andhyperactivity is due to mutation of the second glycine to aserine in thesignature sequence of the Kir subunit GIRK1. Finally, several mutationsin Kir1.1 causing Bartter's syndrome, a renal disorder involvingsalt-wasting, hypokalemia, and metabolic acidosis, are in the sameresidue or similar functional domains as the mutations. Derst, C. et al.(1997) Biochem Biophys Res Commun 230: 641-5; Simon, D. B. et al. (1996)Nat Genet. 14: 152-6. For instance, D74Y in Bartter's syndrome is in theequivalent residue of the D71V mutation. Bartter's mutation W99C andpore mutation V122E (position 150 in Kir2.1) reside in the samefunctional domain as the .DELTA.95-98 and S136F mutations, respectively.A Bartter's mutation is also seen in the C-terminal tail of Kir1.1 atposition 198 (Kir2.1 residue 248) similar to Andersen's R218W, R218Q,G300V, E303K, and .DELTA.314-15.

Electrophysiological data show that WT current is significantly reducedwhen WT and mutant subunits are co-expressed at equal amounts. Thissuggests that only homo-multimers of WT subunits are functional. Thesedata provide evidence against the possibility that the defect is due toaberrant channel co-assembly. If this were not the case, one wouldexpect to see currents from co-injected oocytes at half the wild-typecurrent. The strong dominant negative effects seen in voltage-clampstudies of co-injected subunits indicate that mutant subunits doco-assemble with wild-type subunits. Furthermore, the dominant negativeeffects documented are likely to result from either a disruption inchannel trafficking or channel function once it is expressed in theplasma membrane; these studies cannot distinguish between these twopossibilities. In the case that channels traffic correctly, K.sup.+current inhibition could be due to a number of causes, includingphysical obstruction of the intracellular vestibule or pore, alteredaffinity for K.sup.+ ions, and increased affinity for Mg.sup.2+ orpolyamines that are known to block the pore.

The dominant negative effects on Kir2.1 function have consequences forcardiac and skeletal muscle excitability. LQT is a disorder of cardiacmyocellular repolarization manifested by prolongation of the intervalbetween the onset of ventricular depolarization (upstroke of the QRScomplex) and termination of ventricular repolarization (end of the Twave). Dominantly inherited LQT is due to mutations in the cardiacNa.sup.+ channel (SCN5A) or mutations in subunits encoding the cardiacdelayed rectifier K.sup.+ channels (HERG,KCNQ1, and KCNE1). Wang, Q. etal. (1995) Cell 80: 805-11; Curran, M. E. et al. (1995) Cell 80:795-803;Sanguinetti, M. C. et al. (1995) Cell 81:299-307; Splawski, I. et al.(1997) Nat Genet. 17:338-40; Wang, Q. et al. (1996) Nat Genet. 12:17-23.The common pathophysiological feature of LQT isprolongation of thecardiac action potential due to either enhanced depolarizing current (Nachannel mutations) or reduced repolarizing current (K.sup.+ channelmutations). KCNJ2 is as a novel LQT gene and is important in modulatingcardiac excitability. Previously Kir2.1 was postulated to play animportant, but not exclusive role, in generation of the cardiac inwardrectifier current (I.sub.K1). Nakalmura, T. Y. et al. (1998) Am JPhysiol 274, H892-900; Wible, B. A. et al. (1995) Circ Res 76:343-50.1.sub.K1 contributes significant repolarizing current during theterminal phase of the cardiac action potential and serves as the primaryconductance controlling the diastolicresting membrane potential(E.sub.m) in a trial and ventricular myocytes. Sanguinetti, M. C., &Tristani-Firouzi, M., CARDIAC ELECTROPHYSIOLOGY: FROM CELL TO BEDSIDE,pp. 79-86., (D. P. Zipes, & J. Jalife, eds., W. B. Saunders, 2000).Dominant-negative mutations in Kir2.1 prolong cardiac action potentialsin affected individuals by reducing the amount of repolarizing currentduring the terminal phase. Action potential prolongation creates thesubstrate for early after depolarizations (EADs), the presumptivetrigger for ventricular tachycardia. Tristani-Firouzi, M. et al. (2001)Am J Med 110: 50-9. Whether mutations in Kir2.1 alter the diastolicE.sub.m in cardiomyocytes is not known.

Kir channels play an important, albeit secondary, role in controllingresting Em in skeletal muscle. Horowicz, P. & Spalding, B. C., MYOLOGY:BASIC AND CLINICAL, pp. 405-422 (A. G. Engel, and C. Franzini-Armstrong,eds., McGraw-Hill, Inc. 1994). While Kir2.1, 2.2, and 2.4 are expressedin skeletal muscle, the relative contribution of each subfamily memberis not known. Kubo, Y. et al. (1993) Nature 362, 127-33; Takahashi, N.et al. (1994) J Biol Chem 269: 23274-9; Topert, C. et al. (1998) JNeurosci 18:4096-105. These data identify the importance of Kir2.1 inmodulating skeletal muscle excitability. The reduced resting K.sup.+conductance due to Kir2.1 mutations might allow the unopposed chlorideconductance to shift E.sub.m in the depolarized direction, toward theequilibrium potential of chloride. Depolarization of the cell membranewould inactivate Na.sup.+ channels, which would be unavailable forinitiation and propagation of action potentials. Reduced Na.sup.+current is proposed to be a common feature in the pathophysiology ofhypokalemic periodic paralysis (HypoKPP). Jurkat-Rott, K. et al. (2000)Proc Natl Acad Sci USA 97, 9549-54; Ruff, R. L. (2000) Proc Natl AcadSci USA 97: 9832-3; Ruff, R. L., & Cannon, S. C. (2000) Neurology54:2190-2. Individuals with HypoKPP caused by mutations in the L-typeCa.sup.2+ channel, reduced availability of Na.sup.+ channels ispostulated to be linked to reduced activity of a member of the Kirsuperfamily, the K.sub.ATP channel. Ptacek, L. J. et al. (1994) Cell 77,863-868; Fouad, G. et al. (1997) Neuromuscular Disorders 7: 33-38; Ruff,R. L. (1999) Neurology 53:1556-63; Tricarico, D. et al. (1999) J ClinInvest 103: 675-82. Thus, these data and that of others, support theimportance of Kir channels in modulating skeletal muscle excitability.Ruff, R. L. (1999) Neurology 53:1556-63; Tricarico, D. et al. (1999) JClin Invest 103: 675-82.

Kir2.1 has a significant role in development The importance of ionchannels in the function of muscle, heart, and brain is indisputable asevidenced by the plethora of mutations found associated with justperiodic paralysis, LQT, and epilepsy. However, an intriguing new nichefor ion channels in development has just begun to be recognized,especially with the characterization of the weaver mouse. A mutation inthe pore region of the G-protein coupled inward rectifier potassiumchannel GIRK2 was found to be associated with the defects in neuraldevelopment of weaver. Patil, N. et al. (1995) Nat Genet. 11, 126-9.

These findings in AS and Kir2.1 support this link between ion channelsand developmental signaling. The role of K.sup.+ channels incraniofacial development has not previously been reported. Andersen'sSyndrome and the Kir2.1 knockout mouse both provide evidence that ionchannels play a previously unrecognized role in this process.Developmental characteristics of the Kir2.1 mouse, including narrowingof the maxilla and complete cleft of the secondary palate, provide someintriguing links to the facial dysmorphology seen in many Andersen'spatients. Zaritsky, J. J. et al. (2000) Circ Res 87: 160-6. A study onKir2.1 mRNA expression in rat embryos at embryonic day 12 shows thatKir2.1 mRNA is associated with bone structures in the head, limbs andbody. Karschin, C., & Karschin, A. (1997) Mol Cell Neurosci 10, 131-48.Whether or not Kir2.1 is expressed early enough for craniofacial andother bone morphogenetic events has not been investigated.

There is evidence suggesting that Kir2.1 might be expressed in neuralcrest cells, a population of cells that eventually differentiate intopart of the peripheral nervous system, skin pigment cells, and into thecraniofacial bones. In the promoter region of KCNJ2 there is a putativebinding site for the transcription factor AP-2. Redell, J. B., & Tempel,B. L. (1998) J Biol Chem 273:22807-18. This transcription factor isexpressed predominantly in neural crest cells. Schorle, H. et al. (1996)Nature 381: 235-8; Zhang, J. et al. (1996) Nature 381: 238-41.Investigation of Kir2.1 expression patterns prior to day 12 in mouseembryos could enhance the understanding of the putative role that Kir2.1plays in craniofacial and skeletal morphogenesis. Recent in situhybridization studies have shown that Kir2.1 is expressed in neuralcrest cells of chick embryos. (Data not shown).

Some evidence exists that alterations in KCNJ2 may be responsible inpart for conditions such as fetal wastage and sudden infant deathsyndrome (SIDS). Andersen's Syndrome is an extremely rare disorder.Moreover, there is a high rate of sporadic mutations found in thedisease; as many as one half of alterations may have resulted from asporadic mutation. This evidence indicates that there is strong naturalselection against the Andersen's Syndrome and mutations in KCNJ2.Because Kir2.1 affects the development of many organs and body systems,it may also play a role in fetal wastage. Additionally, because cardiacdysrhythmias have been implicated in SIDS, alterations in KCNJ2 andKir2.1 may also predispose an infant to a risk for SIDS. Accordingly ascreen for alterations in KCNJ2 and Kir2.1 as discussed below, may beused to assess the risk of SIDS or fetal wastage.

Based on the discovery that KCNJ2 gene is responsible for the Andersen'sSyndrome phenotype including a risk for cardiac dysrhythmias, a geneticscreen has been developed for determining if a patient has a heightenedrisk for a cardiac dysrhythmia. The present invention relates to amethod of determining whether a patient has a heightened risk for acardiac dysrhythmia. Because of the variable expression of theAndersen's Syndrome phenotype even within a kindred, the method may beused to diagnose persons who show little or no outward signs ofAndersen's Syndrome, but who still have a high risk of the heartproblems associated with AS. The genetic screen may be used to determineif the patient has an alteration in a single copy of the KCNJ2 gene. Ifthe patient has an alteration in a KCNJ2 gene, the patient may have aheightened risk for a cardiac dysrhythmia as compared to a personwithout an alteration in his or her copies of the KCNJ2 gene. Thealterations in the KCNJ2 gene include mutations and polymorphisms.Included among the mutations are frameshift, nonsense, splice,regulatory and missense mutations. Any method which is capable ofdetecting the mutations and polymorphisms described herein can be used.Such methods include, but are not limited to, DNA sequencing,allele-specific probing, mismatch detection, single strandedconformation polymorphism detection and allele-specific PCRamplification.

KCNJ2 mutations cause increased risk for cardiac dysrhythmia and are thegenetic cause for Andersen's Syndrome. Many different mutations canoccur in KCNJ2. In order to detect the presence of alterations in theKCNJ2 gene, a biological sample such as blood is prepared and analyzedfor the presence or absence of a given alteration of KCNJ2. To detectthe increased risk for cardiac dysrhythmia or for the lack of suchincreased risk, a biological sample is prepared and analyzed for thepresence or absence of a mutant allele of KCNJ2. Results of these testsand interpretive information are returned to the health care providerfor communication to the tested individual. Such diagnoses may beperformed by diagnostic laboratories or, alternatively, diagnostic kitsare manufactured and sold to health care providers or to privateindividuals for self-diagnosis.

The presence of a hereditary condition such as Andersen's Syndrome or anincreased risk for cardiac dysrhythmias may be ascertained by testingany tissue of a human for mutations of the KCNJ2 gene. For example, aperson who has inherited a germine KCNJ2 mutation may be prone todevelop a fatal cardiac dysrhythmia. This can be determined by testingDNA from any tissue of the person's body. Most simply, blood can bedrawn and DNA extracted from the cells of the blood. In addition,prenatal diagnosis can be accomplished by testing fetal cells, placentalcells or amniotic cells formulations of the KCNJ2 gene. Alteration of awild-type KCNJ2, whether, for example, by point mutation or deletion,can be detected by any of the means discussed herein.

There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect sequence variation. Another approachis the single-stranded conformation polymorphism assay (SSCP). Orita M,et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766-2770. This method doesnot detect all sequence changes, especially if the DNA fragment size isgreater than 200 bp, but can be optimized to detect most DNA sequencevariations. The reduced detection sensitivity is a disadvantage, but theincreased throughput possible with SSCP makes it an attractive, viablealternative to direct sequencing for mutation detection on a researchbasis. The fragments which have shifted mobility on SSCP gels are thensequenced to determine the exact nature of the DNA sequence variation.Other approaches based on the detection of mismatches between the twocomplementary DNA strands include clamped denaturing gel electrophoresis(CDGE) (Sheffield V C, et al. (1991) Am. J. Hum. Genet. 49:699-706),heteroduplex analysis (HA) (White M B, et al. (1992) Genomics12:301-306), and chemical mismatch cleavage (CMC) (Grompe M, et al.,(1989) Proc. Natl. Acad. Sci. USA 86:5855-5892). None of the methodsdescribed above will detect large deletions, duplications or insertions,nor will they detect a regulatory mutation which affects transcriptionor translation of the protein. Other methods which might detect theseclasses of mutations such as a protein truncation assay or theasymmetric assay, detect only specific types of mutations and would notdetect missense mutations. A review of currently available methods ofdetecting DNA sequence variation can be found in a review by Grompe(1993). Grompe M (1993) Nature Genetics 5:111-117. Once a mutation isknown, an allele specific detection approach such as allele specificoligonucleotide (ASO) hybridization can be utilized to rapidly screenlarge numbers of other samples for that same mutation. Such a techniquecan utilize probes which are labeled with gold nanoparticles to yield avisual color result. Elghanian R, et al. (1997) Science 277:1078-1081.

A rapid preliminary analysis to detect polymorphisms in DNA sequencescan be performed by looking at a series of Southern blots of DNA cutwith one or more restriction enzymes, preferably with a large number ofrestriction enzymes. Each blot contains a series of normal individualsand a series of LQTS cases. Southern blots displaying hybridizingfragments (differing in length from control DNA when probed withsequences near or including the KCNJ2 locus) indicate a possiblemutation. If restriction enzymes which produce very large restrictionfragments are used, then pulsed field gel electrophoresis (PFGE) isemployed.

Detection of point mutations may be accomplished by molecular cloning ofthe KCNJ2 allele and sequencing the allele using techniques well knownin the art. Also, the gene or portions of the gene may be amplified,e.g., by PCR or other amplification technique, and the amplified gene oramplified portions of the gene may be sequenced.

There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single stranded conformation analysis (SSCP) Orita M, et al.(1989) Proc. Natl. Acad. Sci. USA 86:2766-2770); 2) denaturing gradientgel electrophoresis (DGGE) (Wartell R M, et al. (1990) Nucl. Acids Res.18:2699-2705; Sheffield V C, et al. (1989) Proc. Natl. Acad. Sci. USA86:232-236); 3) RNase protection assays (Filklelstein et al., 1990;Kinszler K W, et al. (1991) Science 251:1366-1370.); 4) allele-specificoligonucleotides (ASOs) (Conner B J, et al. (1983) Proc. Natl. Acad.Sci. USA 80:278-282.); 5) the use of proteins which recognize nucleotidemismatches, such as the E. coli mutS protein (Modrich P (1991) Ann Rev.Genet. 25:229-253); and 6) allele-specific PCR (Ruano G & Kidd K K(1989) Nucl. Acids Res. 17:8392). For allele-specific PCR, primers areused which hybridize at their 3′ ends to a particular KCNJ2 mutation. Ifthe particular mutation is not present, an amplification product is notobserved. Amplification Refractory Mutation System (ARMS) can also beused. Insertions and deletions of genes can also be detected by cloning,sequencing and amplification. In addition, restriction fragment lengthpolymorphism (RFLP) probes for the gene or surrounding marker genes canbe used to score alteration of an allele or an insertion in apolymorphic fragment. Such a method is particularly useful for screeningrelatives of an affected individual for the presence of the mutationfound in that individual. Other techniques for detecting insertions anddeletions known in the art can be used.

In the first three methods (SSCP, DGGE and RNase protection assay), anew electrophoretic band appears. SSCP detects a band which migratesdifferentially because the sequence change causes a difference insingle-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DCGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or in its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof samples. An example of a mismatch cleavage technique is the RNaseprotection method. In the practice of the present invention, the methodinvolves the use of a labeled riboprobe which is complementary to thehuman wild-type KCNJ2 gene coding sequence. The riboprobe and eithermRNA or DNA isolated from the person are annealed (hybridized) togetherand subsequently digested with the enzyme RNase A which is able todetect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the mRNA or gene but can be a segment of either. If theriboprobe comprises only a segment of the mRNA or gene, it will bedesirable to use a number of the se probes to screen the whole mRNAsequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton R G, et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397-440; Shenk T E, et al. (1975) Proc.Natl. Acad. Sci. USA 72:989-993; Novack D F, et al. (1986) Proc. Natl.Acad. Sci. USA 83:586-590. Alternatively, mismatches can be detected byshifts in the electrophoretic mobility of mismatched duplexes relativeto matched duplexes. See, e.g., Cariello N F (1988) Am. J. HumanGenetics 42:726-734. With either riboprobes or DNA probes, the cellularmRNA or DNA which might contain a mutation can be amplified using PCR(see below) before hybridization. Changes in DNA of the KCNJ2 gene canalso be detected using Southern hybridization, especially if the changesare gross rearrangements, such as deletions and insertions.

DNA sequences of the KCNJ2 gene which have been amplified by use of PCRmay also be screened using allele-specific probes. These probes arenucleic acid oligomers, each of which contains a region of the genesequence harboring a known mutation. For example, one oligomer may beabout 30 nucleotides in length, corresponding to a portion of the genesequence. By use of a battery of such allele-specific probes, PCRamplification, products can be screened to identify the presence of apreviously identified mutation in the gene. Hybridization ofallele-specific probes with amplified KCNJ2 sequences can be performed,for example, on a nylon filter. Hybridization to a particular probeunder high stringency hybridization conditions indicates the presence ofthe same mutation in the tissue as in the allele-specific probe.

Nucleic acid analysis via microchip technology is also applicable to thepresent invention. In this technique, thousands of distinctoligonucleotide probes are built up in an array on a silicon chip.Nucleic acid to be analyzed is fluorescently labeled and hybridized tothe probes on the chip. It is also possible to study nucleicacid-protein interactions using these nucleic acid microchips. Usingthis technique one can determine the presence of mutations or evensequence the nucleic acid being analyzed or one can measure expressionlevels of a gene of interest. The method is one of parallel processingof many, even thousands, of probes at once and can tremendously increasethe rate of analysis. Several papers have been published which use thistechnique. Some of the se are Hacia J G, et al. (1996) Nature Genetics14:441-447; Shoemaker D D, et al. (1996) Nature Genetics 14:450-456;Chee M, et al. (1996) Science 274:610-614; Lockhart D J, et al. (1996)Nature Biotechnology 14:1675-1680; DeRisi J, et al. (1996) Nat. Genet.14:457-460; Lipshutz R J, et al. (1995) Biotechniques 19:442-447. Thismethod has already been used to screen people for mutations in thebreast cancer gene BRCA1. Hacia J G, et al. (1996) Nature Genetics14:441-447. The most definitive test for mutations in a candidate locusis to directly compare genomic KCNJ2 sequences from patients with thosefrom a control population. Alternatively, one could sequence messengerRNA after amplification, e.g., by PCR, thereby eliminating the necessityof determining the exon structure of the candidate gene.

Mutations from patients falling outside the coding region of KCNJ2 canbe detected by examining the non-coding regions, such as introns andregulatory sequences near or within the genes. An early indication thatmutations in noncoding regions are important may come from Northern blotexperiments that reveal messenger RNA molecules of abnormal size orabundance in patients as compared to control individuals.

Alteration of KCNJ2 expression can be detected by any techniques knownin the art. These include Northern blot analysis, PCR amplification andRNase protection. Diminished mRNA expression indicates an alteration ofthe wild-type gene. Alteration of wild-type (genes can also be detectedby screening for alteration of wild-type Kir2.1 protein. For example,monoclonal antibodies immunoreactive with Kir2.1 can be used to screen atissue. Lack of cognate antigen would indicate a mutation. Antibodiesspecific for products of mutant alleles could also be used to detectmutant gene product. Such immunological assays can be done in anyconvenient formats known in the art. These include Western blots,immunohistochemical assays and ELISA assays. Any means for detecting analtered Kir2.1 protein can be used to detect alteration of wild-typeKCNJ2 genes. Functional assays, such as protein binding determinations,can be used. In addition, assays can be used which detect Kir2.1biochemical function. Finding a mutant KCNJ2 gene product indicatesalteration of a wild-type KCNJ2 gene.

Mutant KCNJ2 genes or gene products can also be detected in other humanbody samples, such as serum, stool, urine and sputum. The sametechniques discussed above for detection of mutant genes or geneproducts in tissues can be applied to other body samples. By screeningsuch body samples, a simple early diagnosis can be achieved forAndersen's Syndrome or a risk for cardiac dysrhythmia.

Initially, the screening method involves amplification of the relevantKCNJ2 sequence. Alternatively, the screening method involves anon-PCRbased strategy. Such screening methods include two-step labelamplification methodologies that are well known in the art. Both PCR andnon-PCR based screening strategies can detect target sequences with ahigh level of sensitivity.

The most popular method used today is target amplification. Here, thetarget nucleic acid sequence is amplified with polymerases. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). The polymerase chain reaction andother polymerase-driven amplification assays can achieve over amillion-fold increase in copy number through the use ofpolymerase-driven amplification cycles. Once amplified, the resultingnucleic acid can be sequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the target sequences,the biological sample to be analyzed, such as blood or serum, may betreated, if desired, to extract the nucleic acids. The sample nucleicacid may be prepared in various ways to facilitate detection of thetarget sequence; e.g. denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the analytenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniquesknown in the art.

Analyte nucleic acid and probe are incubated under conditions whichpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the analyte. The region of the probeswhich is used to bind to the analyte can be made completelycomplementary to the targeted region of the genes. Therefore, highstringency conditions are desirable in order to prevent false positives.However, conditions of high stringency are used only if the probes arecomplementary to regions of the chromosome which are unique in thegenome. The stringency of hybridization is determined by a number offactors during hybridization and during the washing procedure, includingtemperature, ionic strength, base composition, probe length, andconcentration of formamide. Under certain circumstances, the formationof higher order hybrids, such as triplexes, quadraplexes, etc., may bedesired to provide the means of detecting target sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand which islabeled, either directly or indirectly. Suitable labels, and methods forlabeling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labeledmoiety. A number of the se variations are well known.

As noted above, non-PCR based screening assays are also contemplated inthis invention. This procedure hybridizes a nucleic acid probe (or ananalog such as a methyl phosphonate backbone replacing the normalphosphodiester), to the low level DNA target. This probe may have anenzyme covalently lined to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10.sup.3-10.sup.6increase in sensitivity. For example, the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes are well known.

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digoxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecifically binding the target gene. Allele specific probes are alsocontemplated within the scope of this example.

In one example, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. In a secondexample, the small ligand is recognized by a second ligand-enzymeconjugate that is capable of specifically complexing to the firstligand. A well known embodiment of this example is the biotin-avidintype of interactions. Methods for labeling nucleic acid probes and theiruse in biotin-avidin based assays are well known.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting the gene or genes. Thus, in oneexample to detect the presence of KCNJ2 in a cell sample, more than oneprobe complementary to KCNJ2 is employed and in particular the number ofdifferent probes is alternatively 2, 3, or 5 different nucleic acidprobe sequences. In another example, to detect the presence of mutationsin the KCNJ2 gene sequence in a patient, more than one probecomplementary to KCNJ2 is employed where the cocktail includes probescapable of binding to the allele-specific mutations identified inpopulations of patients with alterations in KCNJ2. In this embodiment,any number of probes can be used.

Large amounts of the polynucleotides of the present invention may beproduced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian or plant or othereukaryotic cell lines. The general methods of purification of nucleicacids are described, e.g., in Sambrook J, et al. (1989) MolecularCloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) or Ausubel F M, et al. (1992) CurrentProtocols in Molecular Biology, (John Wiley and Sons, New York, N.Y.).

The polynucleotides of the present invention may also be produced bychemical synthesis, e.g., by the phosphoramidite method described byBeaucage and Caruthers or the triester method according to Matteucci andCaruthers and may be performed on commercial, automated oligonucleotidesynthesizers. Matteucci M D & Caruthers M H (1981) J. Am. Chem. Soc.103:3185; Beaucage S L, & Caruthers M H (1981) Tetra. Letts.22:1859-1862. A double-stranded fragment may be obtained from thesingle-stranded product of chemical synthesis either by synthesizing thecomplementary strand and annealing the strand together under appropriateconditions or by adding the complementary strand using DNA polymerasewith an appropriate primer sequence.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host may comprise a replication system recognized by thehost, including the intended polynucleotide fragment encoding thedesired polypeptide, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide encoding segment. Expression vectors may include, forexample, an origin of replication or autonomously replicating sequence(ARS) and expression control sequences, a promoter, an enhancer andnecessary processing information sites, such as ribosome-binding sites,RNA splice sites, polyadenylation sites, transcriptional terminatorsequences, and mRNA stabilizing sequences. Such vectors may be preparedby means of standard recombinant techniques well known in the art anddiscussed, for example, in Sambrook J, et al. (1989) Molecular Cloning:A Laboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) or Ausubel F M, et al. (1992) Current Protocols inMolecular Biology, (John Wiley and Sons, New York, N.Y.).

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host, and may include, whenappropriate, those naturally associated with the KCNJ2 or other gene.Examples of workable combinations of cell lines and expression vectorsare described in Sambrook J, et al. (1989) Molecular Cloning: ALaboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) or Ausubel F M, et al. (1992) Current Protocols inMolecular Biology, (John Wiley and Sons, New York, N.Y.); see also,e.g., Metzger D, et al. (1988) Nature 334:31-36. Many useful vectors areknown in the art and may be obtained from such vendors as Stratagene,New England Biolabs, Promega Biotech, and others. Promoters such as thetrp, lac and phage promoters, tRNA promoters and glycolytic enzymepromoters may be used in prokaryotic hosts. Useful yeast promotersinclude promoter regions for met allothionein, 3-phosphoglycerate kinaseor other glycolytic enzymes such as enolase orglyceraldehyde-3-phosphate dehydrogenase, enzymes responsible formaltose and galactose utilization, and others. Appropriate non-nativemammalian promoters might include the early and late promoters from SV40or promoters derived from murine Molony leukemia virus, mouse tumorvirus, avian sarcoma viruses, adenovirs II, bovine papilloma virusorpolyoma. Insect promoters may be derived from baculovirus. Fiers W, etal. (1978) Nature 273:113-120. In addition, the construct may be joinedto an amplifiable gene (e.g., DHFR) so that multiple copies of the genemay be made. For appropriate enhancer and other expression controlsequences, see also Enhancers and Eukaryotic Gene Expression, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1983).

While such expression vectors may replicate autonomously, they may alsoreplicate by being inserted into the genome of the host cell, by methodswell known in the art.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc., b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribedinvitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection (see, Kubo T, et al. (1988) FEBSLett. 241:119), or the vectors can be introduced directly into hostcells by methods well known in the art, which vary depending on the typeof cellular host, including electroporation; transfection employingcalcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, orother substances; microprojectile bombardment; lipofection; infection(where the vector is an infectious agent, such as a retroviral genome);and other methods. See generally, Sambrook J, et al. (1989) MolecularCloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) and Ausubel F M, et al. (1992) CurrentProtocols in Molecular Biology, (John Wiley and Sons, New York, N.Y.).The introduction of the polynucleotides into the host cell by any methodknown in the art, including, inter alia, those described above, will bereferred to herein as “transformation.” The cells into which have beenintroduced nucleic acids described above are meant to also include theprogeny of such cells.

Large quantities of the nucleic acids and polypeptides of the presentinvention may be prepared by expressing the KCNJ2 nucleic acid orportions thereof in vectors or other expression vehicles incompatibleprokaryotic or eukaryotic host cells. The most commonly used prokaryotichosts are strains of Escherichia coli, although other prokaryotes, suchas Bacilluis subtilis or Pseudonmonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby W B and Pastan I H (eds.) (1979) Cell Culture. Methods inEnzymology volume 58 (Academic Press, Inc., Harcourt Brace Jovanovich(New York)). Examples of commonly used mammalian host cell lines areVERO and HeLa cells, Chinese hamster ovary (CHO) cells, and W138, B, andCOS cell lines, although it will be appreciated by the skilledpractitioner that other cell lines may be appropriate, e.g., to providehigher expression, desirable glycosylation patterns, or other features.An example of a commonly used insect cell line is SF9.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule,preferably the same DNA molecule. In prokaryotic hosts, the transformantmay be selected, e.g., by resistance to ampicillin, tetracycline orother antibiotics. Production of a particular product based ontemperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides ofthe present invention will be useful not only for the production of thenucleic acids and polypeptides of the present invention, but also, forexample, in studying the characteristics of Kir2.1 or otherpolypeptides.

The mutated genes and their encoded proteins may be used in methods ofdiagnosing a patient with Andersen's Syndrome or to further study theproperties of Kir2.1. KCNJ2 genes with an alteration such as ainsertion, a deletion, a missense mutation are presented herein. Suchaltered KCNJ2 genes may have the sequence of SEQ ID NO 1 as altered byone or more mutations selected from the group consisting of A440T,G658A, A874C, C880T, G1127T, G1132A, C635T, G881A, G1135A, C785T, C452G,G439A, a 6 nucleotide deletion beginning with nucleotide 1167, and a 12by deletion beginning with nucleotide 512. Altered Kir2.1 polypeptideswhich are encoded by altered KCNJ2 genes have been isolated, purifiedand sequenced. For example, Kir2.1 proteins have been isolated whichhave alterations in highly conserved positions within the Kir2.1protein. Examples of such proteins are proteins which have the aminoacid sequence of SEQ ID NO 2 as altered by one or more of the followingalterations: D71V, G144S, N216H, R218W, G300V, V302M, .DELTA.314-315,.DELTA.95-98, S136F, R218Q, E303K, P186L, T75R, and D71N.

In order to better describe the details of the present invention, thefollowing discussion is divided into six sections: (1) patients withAndersen's Syndrome have variable expressivity; (2) an Andersen'sSyndrome allele is located on chromosome 17q23; (3) KCNJ2, CACNG1 andSCN4A are candidate genes for Andersen's Syndrome; (4) D71V segregateswith Andersen's Syndrome; (5) thirteen additional mutations have beenidentified in Andersen's Syndrome probands; and (6) D71V and R218Wresult in a dominant negative effect on Kir2.1 current in Xenopusoocytes.

Patients with Andersen's Syndrome have Variable Expressivity

There are no published clinical criteria for the diagnosis of Andersen'sSyndrome. The criteria based upon the clinical data gathered from thethree largest published case series on Andersen's Syndrome, a set ofcriteria were developed. Canun, S., et al. (1999) Am J Med Genet. 85:147-56; Sansone, V. et al (1997) Ann Neurol 42: 305-12; Tawil, R. et al.(1994) Ann Neurol 35:326-30. These criteria take into account theclinical observation that Andersen's Syndrome shows variable penetrance.

Individuals were classified as affected if two of three of the followingcriteria were met: clearcut episodes of muscle wellness, cardiacinvolvement, and dysmorphology. Muscle weakness was based on one of thefollowing criteria: 1) a typical history of weakness with rest followingexertion or prolonged rest, 2) an a typical history but with adocumented physical exam during an attack demonstrating hypo reflexiawith preserved sensation, 3) an atypical history without a documentedexam but with unexplained intraictal serum hypo/hyperkalemia, or 4) anatypical history without a documented exam or serum potassium levels butwith an abnormal exercise nerve conduction study. McManis, P. G. et al.(1986) Muscle Nerve 9, 704-10. Cardiac involvement was determined by thepresence of prolonged QTc on twelve lead EKG according to standardcriteria. Martin, A. B. et al. (1995) Am J Cardiol 75, 950-2 Schwartz,P. J. et al. (1993) Circulation 88: 782-4. Dysmorphology was noted ifthere was the presence of two or more of the following: 1) low set ears,2) hypertelorism, 3) small mandible, 4) clinodactyl), or 5) syndactyl).At risk individuals expressing one of the three major phenotypes ofAndersen's Syndrome were classified as “probably affected”. Individualswere classified as unaffected if none of the criteria were fulfilled.One of the authors (R. T.), who was blinded to the results of themutational analysis, reviewed the clinical information on each subjectand confirmed their diagnostic classification.

A total of 32 unrelated Andersen's Syndrome kindreds were identifiedmeeting the defined diagnostic criteria. Fourteen different mutationswere found in the KCNJ2 gene of 22 out of the 32 kindreds. Most kindredswere small, consisting of one to three affected individuals. Affectedindividuals showed marked variability in the phenotypic expression ofthe disease. Whereas one individual, typically the index case,manifested the full Andersen's triad, other affected individualsdemonstrated only one or two of the major characteristics of thisdisorder. Dysmorphic features ranged from negligible deformities of thedigits to very prominent facial dysmorphisms. Cardiac involvement rangedfrom a symptomatic LQT and ventricular ectopy, to syncope from sustainedventricular tachycardia, to recurrent torsades depointes and cardiacarrest requiring treatment with an implantable defibrillator. Attacks ofparalysis were associated with hypo-, hyper-, or normokalemia althoughserum potassium levels during attacks differed among kindreds they wereconsistent within an individual kindred.

The expressivity of Andersen's Syndrome is variable. Andersen's Syndromeis incompletely penetrant and variably expressed. Severity ranges fromnon-penetrant (4 of 28 affected individuals), 1 of 3 characteristics (5of 28), 2 of 3 characteristics (6 of 28), to severely affected with 3out of 3 characteristics (13 of 28). Several conditions could explainsuch pleiotropy. First, normal variation in mutant versus wild-typesubunit expression levels could explain some of this variation. It wasshown in electrophysiological studies with the weaver mutation thatlower levels of mutant subunit in comparison to wild-type reducedchannel function. However, when levels of mutant subunits wereincreased, these K.sup.+-specific channels began to aberrantly passNa.sup.+ current. Slesinger, P. A. et al. (1996) Neuron 16: 321-31. Inaddition to mutant subunit expression levels, normal variations inexpression of overlapping channels or in non-channel proteins within thesame functional pathway as Kir2.1 could cause phenotypic variation.External environmental factors could also have some influence on theexpression of the Andersen's phenotypes. Because of the variableexpressivity, it is likely that some Andersen's patients are diagnosedas LQT or periodic paralysis patients instead.

An Andersen's Syndrome Allele is Located on Chromosome 17q23

In order to identify the Andersen's locus, approximately 400 polymorphicmarkers were analyzed across the entire genome in 15 individuals ofkindred 4415 (FIG. 1A). Kindred 4415 is well-established as an ASfamily, individuals were classified as affected in this analysis if theyexhibited one of the three main characteristics of Andersen's Syndromeas described previously. One marker (D175949) from the set of 400maximized at .theta.=0 with a LOD score of 3.23. The simulated maximumLOD score for this kindred for a 5 allele system was 3.21 at .theta.=0.Marker D175787 set the proximal recombinant boundary with a LOD scoreof-.infin. at .theta.=0, whereas D17S784 set the distal recombinantboundary with a LOD score of-.infin. at .theta.=0. This regioncorresponds to a genetic region of over 40 cM on chromosome 17q23 (FIG.3).

An automated genome-wide screen was performed on fifteen individuals inkindred 4415 (FIG. 2A) using the ABI marker index of 400 polymorphicmarkers. Markers were distributed across the genome at .about.10-20 cMintervals. The fluorescently labeled markets were used to amplifygenomic DNA in total reaction volumes of 20.mu.l in an MJRPTC-200thermocycler (MJ Research). The products were visualized on an AppliedBiosystems Model 373A and analyzed by the Genotyper peak-callingsoftware. Pairwise linkage analysis was performed using the MLINKprogram of the LINKAGE package. Lathrop, G. M. et al. (1985) Am J HumGenet. 37, 482-98. Disease penetrance was set at 0.95 without a genderdifference, and the normal and disease allele frequencies were set at0.999 and 0.001, respectively.

KCNJ2, CACNG1 and SCN4A are Candidate Genes for Andersen's Syndrome

The region of chromosome 17q23 defined by the obligate recombinantboundaries of proximal marker D175787 and distal marker D175784 wasexamined in the map viewer database (http://www.ncbi.nlm.nihgov/genone/g-uide). Candidate genes were selected based on theirlocation within these boundaries as ascertained from the availablephysical map. The entire coding region of Kir2.1 was amplified(.about.1.6 kb) from genomic DNA in all individuals from kindred 4415.PCR primer sequence is as follows: F1 5′ CCAAAGCAGAAGCACTGGAG3′ (SEQ IDNO.: 3) and R1 5′ AATCAAATACCCAACCAAGGC 3′ (SEQ ID NO.: 4). 50.mu.l PCRreactions were performed on 100 ng of genomic DNA and 20 μmol of each F1and R1PCR primers using Clonetech's Advantage-GC cDNA polymerase andbuffers. The GC-meltmix was used at a final concentration 1.0 mM, andreactions were cycled under the following protocol: 94.degree. C.-2 min,(94.degree. C.-10 sec, 60.degree. C.-20 sec, 68.degree. C.-2min).times.45, 68.degree. C.-2 min and 30 sec, 4.degree. C.-hold. Theseproducts were prepared using the Qiaquick PCR spin prep kit (Qiagen) andwere sequenced using the following primers: F1, R1, F2 5′GTGTTTGATGTGGCGAGTGG 3′ (SEQ ID NO.: 5) and R25′ ATTCCACTGTCAAACCCAAC 3′(SEQ ID NO.: 6). Sequencing was performed by the Core facility at theUniversity of Utah. Substitution mutations were checked in over 100unaffected unrelated individuals either by SSCP analysis or bymutation-specific PCR analysis (See Table 1). For these individuals,genomic DNA was PCR amplified using the above protocol. These reactionswere then diluted in 100.mu.l of water and served as templates withwhich to perform SSCP analysis or mutation-specific PCR (MSP) analysis.For SSCP and MSP, 2.mu.l of diluted PCR reaction was used as templateDNA. SSCP was performed with 10.mu.l reactions as described previously.Ptacek, L. J. et al. (1991) Cell 67, 1021-7. Products wereelectrophoresed according to Table 1 and visualized using standardtechniques. MSP analysis was performed on four mutations that could notbe visualized using SSCP. Individuals were PCR amplified using eitherthe forward (F) and reverse (R) control primers or the forward mutant(M) primer and the reverse control primer. Products were electrophoresedside-by-side on a 1% agarose gel.

Previous findings that periodic paralysis and LQT are associated withmutations in ion channels led to the prediction that AS might also bedue to this same mechanism. Chromosome 17q23 contigs contained three ionchannel genes within the linked region, KCNJ2, calcium channel CACNG1,and sodium channel SCN4A (FIG. 3). These channels are expressed inskeletal muscle and heart. Two findings led to focusing on KCNJ2: 1) anon-obligatere combination between the most highly linked marker andCACNG1 and SCN4A in kindred 4415 (data not shown) was identified, and 2)SCN4A had already been shown to be responsible for periodic paralysiswithout heart or developmental problems. Ptacek, L. J. et al. (1991)Cell 67:1021-7. Due to the known function and expression pattern ofKir2.1,KCNJ2 is an excellent candidate gene for Andersen's Syndrome.

D71V Segregates with Andersen's Syndrome

The coding region of KCNJ2, contained within one exon, was PCR amplifiedand sequenced in all individuals from kindred 4415 from whom DNA wasavailable. This excludes the deceased individual in the firstgeneration. An A to T transversion corresponding to the mutation D71Vwas identified in all affected individuals but not in any unaffectedfamily members (FIGS. 1A and 1B). DNA from over 100 unaffected unrelatedindividuals was examined for this base pair change usingmutation-specific PCR, and the change was never observed. This residueis absolutely conserved not only between human and rodent, but also inall identified families of the inward rectifier K.sup.+ subunits (FIG.1C). This mutation lies in the cytoplasmic N-terminal segment of Kir2.1(FIG. 1B) in a region of unknown function.

Additional Mutations have been Identified in Andersen's SyndromeProbands

Subsequently thirty-one additional unrelated AS probands (FIG. 4) formutations in KCNJ2 were examined. As with kindred 4415, the codingregion of KCNJ2 was PCR amplified and sequenced. In total, fourteenmutations were identified in twenty-two probands (FIGS. 4 and 5). Threeof the sixteen families examined do not have any mutations in the codingregion of KCNJ2. No discernible difference was observed in the clinicalmanifestations between kindreds with Kir2.1 mutations and those withouta definable mutation. The mutations occur at highly conserved residues(FIG. 5) and include: D71V, .DELTA.95-98, S136F, G144S, R218W, R218Q,G300V, E303K, and .DELTA.314-15. Twelve of the fourteen mutations aremissense mutations consisting of the following changes (Genbankaccession #AF153819): D71V (A440T), S136F (T635C), G144S (G658A), R218W(C880T),R218Q (G881A), G300V(G1127T), E303K(G1135A), N216H (A874C),V302M (G1132A), P186L (C785T), C52G (T75R), and D71N (G439A). Twomutations are in-frame deletions: .DELTA.95-98 (bp 513-524) and.DELTA.314-15 (bp 1167-1172). All substitution mutations were checked inover 100 unaffected unrelated individuals by SSCP or mutation-specificPCR analysis (Table 1) and were never seen in this panel. R218W occurredin five families, and G300V is present in two families. All othermutations were only identified in single families. At least three of thechanges represent de novo mutations (G144S, R218W (three events), and.DELTA.314-15). Only one polymorphism was identified in AS probands.This polymorphism is a silent mutation of C1374T in the codon forresidue L382.

TABLE 1 Primer sequences and mutational analysis conditions. “T.sub.a”refers to the PCR annealing temperature. Visualization of PCR productswas by 1% agarose gel electro-phoresis except where denoted by an “*”next to the mutation. These exceptions were visualized using standardSSCP techniques. See “Mutational analysis” in the experimental methodssection. Mutation Primer sequence Method T.sub.a D71V F 5′GGCAACGGTACCTCGCAGA 3′ SEQ ID. NO.: 7 MSP 62.degree. C. M 5′GGGAACGATACCTCGCAGT 3′ SEQ ID. NO.: 8 R 5′CAACCAAAACACACAGCCAAA 3′ SEQID. NO.: 9 S136F F 5′ CGGCTGCCTTCCTCTTCTC 3′ SEQ ID. NO.: 10 MSP64.degree. C. M 5′ CGGGTGCCTTCCTCCTCTT 3′ SEQ ID. NO.: 11 R5′GTTTCTCTTCTTTGGCTTTGC 3′ SEQ ID. NO.: 12 G144S*F 5′ GCTTCACGGCTGCCTTCC3′ SEQ ID. NO.: 13 SSCP 55.degree. C. R 5′ GTTTCTCTTCTTTGGCTTTGC 3′ SEQID. NO.: 14 R218Q F 5′ GGCGAGTGGGCAATCTTCG 3′ SEQ ID. NO.: 15 MSP62.degree. C. M 5′GGCGAGTAGGCAGTCTTCA 3′ SEQ ID. NO.: 16 R 5′CTCAAATCATATAAAGGACTGTC 3′ SEQ ID. NO.: 17 R218W F 5′GGCGAGTGGGCAATCTTCG 3′ SEQ ID. NO.: 18 MSP 57.degree. C. M 5′GTCGCGAGTAGGCAATGTTT 3′ SEQ ID. NO.: 19 R 5′ CTCAAATCATATAAAGGACTGTC 3′SEQ ID. NO.: 20 G300V*F 5′ AGGACATTGACAACGCAGAC 3′ SEQ ID. NO.: 21 SSCP55.degree. C. R 5′CATGGCAGTGGCTTCCACC 3′ SEQ ID. NO.: 22 E303K F 5′CATACTGGAAGGCATGGTGG 3′ SEQ ID. NO.: 23 MSP 63.degree. C. M 5′CATGCTGGAAGGAATGGTGA 3′ SEQ ID. NO.: 24 R 5′ GTTTTGTGGAACCTGGAATAG 3′SEQ ID. NO.: 25

The mutations are located throughout the protein (FIG. 2B). D71V residesin the N-terminus in the last position of a predicted alpha-helix. Thisresidue is just C-terminal to the putative N-terminal interactiondomain. Tucker, S. J., & Ashcroft, F. M. (1999) J Biol Chem 274:33393-7. Deletion .DELTA.95-98 removes four residues of the M1transmembrane segment. This might completely prohibit the M1 segmentfrom being inserted into the membrane. S136F and G144S are both poremutations. G144S is located in the first position of the highlyconserved K.sup.+ channel signature sequence GYG. Several mutationsreside in the C-terminus. R218W and R218Q are located within theC-terminal interaction domain, and G300V, E303K, and .DELTA.314-15 arelocated in a region without a described function. Tinker, A. et al.(1996) Cell 87: 857-68.

D71V and R218W Result in a Dominant Negative Effect on Kir2.1 Current inXenopus Oocytes

The ability of mutant Kir2.1 subunits to form functional homomultimericchannels was assessed by comparing oocytes injected with wild-type (WT)or mutant Kir2.1 CRNA (23 ng/oocyte). Injection of WT Kir2.1 inducednearly instantaneous K.sup.+ currents that demonstrated strong inwardrectification (FIGS. 6A and 7A), as previously described. Raab-Grahan,K. F. et al. (1994) Neuroreport 5, 2501-5. Inward rectification refersto the property that permits inward flux of K.sup.+ ions at potentialsnegative to the K.sup.+ equilibrium potential (E.sub.K) more readilythan outward flux at positive potentials. At extreme hyper polarizedpotentials, the currents decayed due to voltage and time-dependentblockade by external Na.sup.+ ions. Biermans, G. et al. (1987) PflugersArch 410:604-13. D71V and R218W mutant subunits failed to formfunctional homomultimeric channels. Injection of D71V or R218W cRNA didnot induce detectable K.sup.+ currents (FIGS. 6B and 7B). Smallendogenous currents, identical to those in H.sub.20-injected controloocytes, were recorded in oocytes injected with mutant cRNA.

As is an autosomal-dominant disorder and, as such, affected individualspossess one normal and one mutant KCNJ2 allele. To assess the ability ofmutant Kir2.1 subunits to form functional heteromultimeric channels withWT subunits, mutant (11.5 ng/oocyte) and WT Kir2.1 cRNA (11.5 ng/oocyte)were co-injected and compared currents to those induced by injection ofWT Kir2.1 cRNA (23 ng/oocyte). Co-expression of WT and D71 V Kir2.1induced an inwardly rectifying K current whose current amplitude wasmarkedly reduced (FIGS. 6B and 6C). Current amplitude at −150 mV was−4.61.+−0.37.mu.A for WT Kir2.1, compared to −0.26.+−0.02.mu.A forco-injected WT and D71V. Assuming random association of WT and mutantKir2.1 subunits, 1/16 of the channels will be comprised of four WTsubunits, whereas 15/16 of channels will contain one or more mutantsubunits. The reduction in current induced by co-expression of WT andD71V subunits was approximately 15/16 that of WT current, suggestingthat one mutant D71V subunit is sufficient to eliminate channelfunction. Co-expression of WT and R18W Kir2.1 also induced inwardlyrectifying K.sup.+ currents, although the magnitude of current reductionwas not as severe as that seen with D71V subunits (FIGS. 7B and 7C).These findings demonstrate that D71V and R218W subunits co-assemble withWT Kir2.1 subunits and cause variable degrees of dominant-negativesuppression of channel function.

SUMMARY

In summary, mutations in the Kir2.1 gene, KCNJ2, are responsible forAndersen's Syndrome. Humans affected with Andersen's Syndrome have ahigh risk for fatal cardiac dysrhythmias. The phenotypic expression ofAndersen's Syndrome varies widely with some affected individualspotentially going undiagnosed. Thus, a method of diagnosing Andersen'sSyndrome is presented. The method uses a genome screen for mutations inKCNJ2. Moreover, similar screens are provided which may determine if anindividual seemingly unaffected by Andersen's Syndrome or other long QTdisorders may in fact have a heightened risk for a potentially fatalcardiac dysrhythmia.

The present invention may be embodied in other specific forms withoutdeparting from its structures, methods, or other essentialcharacteristics as broadly described herein and claimed hereinafter. Thedescribed embodiments are to be considered in all respects only asillustrative, and not restrictive. The scope of the invention is,therefore, indicated by the appended claims, rather than by theforegoing description. All changes that come within the meaning andrange of equivalency of the claims are to be embraced within theirscope.

1.-30. (canceled)
 31. A method of detecting a risk of KCNJ2 genemutation associated cardiac dysrhythmia in a human subject who has nooutward signs of Andersen's Syndrome, comprising: a) obtaining abiological sample from the subject; b) preparing a copy of a KCNJ2 genefrom the sample; c) screening for A440T, T635C, G658A, C880T, G881A,G1127T, and G1135A alterations in the copy of the KCNJ2 gene; d)identifying the presence or absence of an alteration, and e) assessingthe subject's risk of cardiac dysrhythmia associated with mutations inthe KCNJ2 gene based on the presence or absence of an alteration in thesubject's KCNJ2 gene, wherein if an alteration in the KCNJ2 gene isdetected the subject is assessed an increased risk for cardiacdysrhythmia.
 32. The method of claim 31, wherein the alteration in thecopy of the KCNJ2 gene is selected from the group consisting of: amissense mutation, a deletion, an in-frame deletion, and an insertion.33. The method of claim 31, wherein the identification the presence orabsence of a KCNJ2 gene alteration includes assaying and analyzing thesample utilizing at least one technique selected from the groupconsisting of: Northern blot analysis, PCR amplification, RNaseprotection, monoclonal antibodies, Western blots, and ELISA assay. 34.The method of claim 31, wherein the human subject is an infant.
 35. Amethod of detecting a risk of KCNJ2 gene mutation associated suddeninfant death syndrome in a human infant subject comprising: a) obtaininga biological sample from the subject; b) preparing a copy of a KCNJ2gene from the sample; c) screening for A440T, T635C, G658A, C880T,G881A, G1127T, and G1135A alterations in the copy of the KCNJ2 gene; d)identifying the presence or absence of an alteration, and e) assessingthe subject's risk of sudden infant death associated with mutations inthe KCNJ2 gene based on the presence or absence of an alteration in thesubject's KCNJ2 gene, wherein if an alteration in the KCNJ2 gene isdetected the subject is assessed to have an increased risk for suddeninfant death.
 36. The method of claim 35, wherein the alteration in thecopy of the KCNJ2 gene is selected from the group consisting of: amissense mutation, a deletion, an in-frame deletion, and an insertion.37. The method of claim 35, wherein the assaying and analyzing of thesample is accomplished utilizing at least one technique selected fromthe group consisting of: Northern blot analysis, PCR amplification,RNase protection, monoclonal antibodies, Western blots, and ELISA assay.